PROJECT SUMMARY Mesenchymal stem cells (otherwise known as mesenchymal stromal cells or MSCs) hold great promise for treatment of bone related disorders due to their paracrine, multi-potential and immunosuppressive effects. One major objective is to leverage the osteogenic differentiation of MSCs as potential treatments in metabolic bone disorders (e.g. osteoporosis, osteogenesis imperfecta, or hypophosphatasia) or localized bone defects. However, knowledge of the MSC differentiation control pathways and how to leverage these during bioprocessing is lacking and has impeded more widespread clinical translation1-3. Unfortunately, the current clinical and industry state-of-the-art for characterization of MSC potency is to simply measure a few surface markers using flow cytometry and to study the ability of cells to differentiate in vitro into the target tissues (e.g. bone/cartilage). These assays are challenging to quantify, slow to perform, and have low direct relevance to in vivo functioning4. Our proposal aims to leverage development of novel imaging technologies to identify in vivo processes of key significance to transplanted stem cell differentiation and healthy bone tissue production, and will interrogate and leverage membrane lipid pathways to maintain and enhance the differentiation potential of MSCs. Our planned research is motivated by growing appreciation of how lipid membrane processing regulates cellular properties of central regulatory importance in bone formation, where maturing osteoblasts produce and secrete mineralizing matrix vesicles in multivesicular bodies of ~500 nanometer diameter that nucleate the deposition and growth of hydroxyapatite mineral crystals in bone collagen5,6. Little is known about processes that drive the formation and secretion of these vesicles, which is a pathway of central importance in the formation of mature cartilage and bone. This formation process is challenging to label, but with a high refractive index change it is ideal for refractive index sensitive modalities like third harmonic generation imaging. In this work, we hypothesize that 3-dimensional time-resolved super resolution membrane imaging will provide a direct readout of vesicle formation profiles that will predict cell differentiation and potency. In these studies, we will develop ultra-resolution multiphoton microscopy to monitor MSC collagen production and mineralizing vesicle release during osteogenic differentiation. We will then apply this technology in vivo using adaptive scattering wavefront correction for intravital super-resolution third harmonic generation microscopy to monitor MSC matrix vesicle formation and produce high potency osteogenic cells. This will significantly advance scientific understanding from a technological perspective by enabling the label- free super-resolution 3-dimensional visualization of nanometer-sized features through completely opaque highly scattering bone tissue. We have the potential to transform treatment of bone diseases, with results that would be broadly applicable to a range of human health concerns.